The overall objective of this revised R21 application is to determine the potential of mouse retinal progenitor cells (RPCs) to differentiate into retinal ganglion cells (RGCs) outside of their normal embryonic environment. The potential of embryo- or embryonic stem cell-derived RPCs to differentiate into functional RGCs in adult retinas will be examined in vivo by transplanting them into adult host retinas in which RGCs have been genetically ablated. A novel genetic mouse model has been developed in which some or all RGCs can be ablated at any time during adult life with the consequent degeneration of the optic nerve. Retinas from these mice provide a unique microenvironment for cell transplantation approaches to restore RGCs and regenerate the optic nerve or prevent its further degeneration. The experimental plan depends on understanding the key regulatory events that control the specification and differentiation of RGCs during retinogenesis. The proneural basic helix-loop-helix factor Math5 occupies a central node in the gene regulatory network that controls RGC development because it is responsible for endowing RPCs with the competence to acquire a RGC fate. The hypothesis to be tested is that Math5-expressing RPCs are a distinct, isolatable RPC subpopulation and that Math5-expressing RPCs will retain their developmental potential even when placed into the microenvironment of the adult retina. To investigate the interactions between Math5-expressing RPCs and the adult retinal microenvironment, the potential of purified Math5-expressing RPCs to differentiate into functional RGCs after transplantation into RGC- ablated adult retinas will be investigated. Determining the developmental potential of embryonic RPCs to adapt to the microenvironment of the adult retina will contribute towards an understanding of retina development as well as establishing more robust methods to prevent, repair and regenerate damaged retinas. Genetic ablation of RGCs in adult mice provides a new model for retinal regeneration. Knowledge of the mechanisms that allow RPCs to differentiate into RGCs in their normal environment provides the underpinning for generating large numbers of functional RGC progenitors from embryonic retinas or pluripotent embryonic stem cells. PUBLIC HEALTH RELEVANCE: This project will reveal new insights into the properties of the embryonic retinal progenitor cells that give rise to retinal ganglion cells in the adult neural retina. Experiments are designed to determine the potential of retinal progenitor cells to adapt to the microenvironment of the adult retina rather than their normal embryonic microenvironment. The project will contribute to an understanding of retinal development as well as establishing new approaches to regenerate damaged retinas and prevent or repair optic nerve degeneration in retinal pathologies such as optic neuritis, ischemic optic neuropathy, and glaucoma.